The activation of phosphatidylinositol-hydrolyzing phospholipase A2 during prostaglandin synthesis in transformed mouse BALB/3T3 cells.
To investigate the type of phospholipase activated by agents that stimulate prostaglandin synthesis, we used transformed mouse cells whose phospholipids were doubly labeled with [14C]inositol and [3H]arachidonic acid. [14C]Inositol was incorporated mostly into the phosphatidylinositol and [3H]arachidonic acid was distributed into the various phospholipids. When these cells were incubated with bradykinin, a stimulator of prostaglandin synthesis, the release of 3H radioactivity from cellular phospholipids and the synthesis of prostaglandin were initiated within seconds and reached a maximum in 40 to 70 s. Analysis of the intracellular lipids revealed a concomitant increase of radioactivity associated with lysophosphatidylinositol, which was detectable within 5 s of incubation with bradykinin and reached a maximum between 40 and 70 s. Lysophosphatidylinositol which could be formed either from a phospholipase A1 or phospholipase A2 reaction, was identified by its chromatographic properties and conversion to glycerophosphorylinositol. We found that the 3H/14C ratio of purified lysophosphatidylinositol was 1/11 of that of phosphatidylinositol, which indicated that lysophosphatidylinositol formed in response to bradykinin is 1-acyl-sn-glycero-3-phosphorylinositol and most probably is formed from a phospholipase A2 deacylation of phosphatidylinositol (a phospholipase A1 deacylation would result in the formation of lysophosphatidylinositol of a 3H/14C ratio similar to phosphatidylinositol). Furthermore, we did not detect between control and stimulated cells any significant difference in the level of several phospholipase C metabolites including inositol phosphate, diglyceride, and phosphatidic acid. These results suggest that phospholipase C is probably not activated. The formation of lysophosphatidylinositol was also stimulated by thrombin and ionophore A23187, both activators of prostaglandin synthesis. Dexamethasone, a lipase inhibitor, inhibited the appearance of lysophosphatidylinositol, whereas aspirin and low concentrations of indomethacin, the cyclooxygenase inhibitor, did not inhibit. The results presented in ths paper provide evidence that a phospholipase A2-hydrolyzing phosphatidylinositol is activated when intact cells are stimulated for prostaglandin synthesis.
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054466:Phospholipases A1; D054467:Phospholipases A2; D011453:Prostaglandins / Q000096:biosynthesis*; D011458:Prostaglandins E / Q000096:biosynthesis; D013917:Thrombin / Q000494:pharmacology
D010716:Phosphatidylinositols; D011453:Prostaglandins; D011458:Prostaglandins E; D000001:Calcimycin; D010740:Phospholipases; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D013917:Thrombin; D001920:Bradykinin